The aim of this study was to investigate the influence of different assessment time periods of submaximal tests on the determination of the maximal accumulated oxygen deficit (MAOD), through the adoption of different time slots of 4 to 6, 6 to 8 and 8 to 10 min. Ten cyclists with mean age of 27.5 ± 4.1 years, body mass 74.4 ± 12.7 kg and time experience of 9.8 ± 4.7 years participated in this study. The athletes underwent an incremental exercise test to determine the peak oxygen consumption (VO2peak), and four submaximal constant work-load test sessions (60, 70, 80 and 90% VO2peak) of 10 min in order to estimate the O2 demand (DEO2). The mean VO2 values obtained on each constant work-load for the 4 to 6, 6 to 8 and 8 to 10 min time-periods intervals were used to perform a linear regression between the intensity and O2 consumption for each time-period. In addition, the subjects performed one supramaximal rectangular test (110% VO2peak) for the quantification of MAOD. There was no significant difference in VO2 between the different time-periods for all submaximal tests (P> 0.05). Similarly, no significant difference was found in DEAO2 and MAOD (P> 0.05). Furthermore, the values of MAOD for the three time-periods intervals showed good agreement and strong correlation. Thus, the data suggest that the submaximal tests used to estimate the values of MAOD can be reduced, at least in this type of sample, and with the use of a cycle simulator.